The oral/gastrointestinal, nasopharyngeal, respiratory, and vaginal mucosae are natural sites for entry of many serious human pathogens. To protect against those pathogens, it is necessary to evoke strong pathogenspecific, protective mucosal immune responses. Endogenous immunoregulatory systems on the mucosae, however, routinely suppress immune responses to most antigens (Ag) at those sites. LT-IIb, a Type II heatlabile enterotoxin (HLT) of Escherichia coli, is a potent mucosal immunostimulant (adjuvant) which, when co-administered with Ag, alters or bypasses those regulatory systems to dramatically enhance mucosal and systemic immune responses to foreign Ags. Upon intranasal administration, LT-IIb exerts immunostimulatory effects using immune pathways and cellular receptors different from those employed by cholera toxin (CT), LT (LT-I) of E. coli, and other mucosal adjuvants. We have shown that activation of these distinctive pathways is modulated by binding of LT-IIb to specific ganglioside receptors located on the surface of immunocompetent cells. Recently, we demonstrated that LT-IIb(T13I), a mutant of LT-IIb with altered ganglioside binding affinities, exhibits no toxic activity, yet retains all of the immunostimulatory properties of the parental HLT. Using a novel knock-out mouse (cmah-/-) which mimics the ganglioside-expression pattern of humans (never employed previously to study HLT adjuvanticity), we showed that intranasal administration of LT-IIb(T13I), in contrast to LT-IIb or CT, does not evoke inflammatory cytokines in the brain, an undesirable response elicited by CT, LT-I, and LT-IIb. We also demonstrated that the B pentamer of LT-IIb (LT-IIb-B5), the non-toxic, ganglioside-biding moiety of LT-IIb, also exhibits potent mucosal adjuvant activity. Yet, unlike LT-IIb holotoxin or LT-IIb(T13I) holotoxin, the immunostimulatory properties of LT-IIb- B5 depend upon expression of by immunocompetent cells of TLR2. Thus, LT-IIb(T13I) and LT-IIb-B5 utilize independent immunostimulatory mechanisms. We hypothesize, therefore, that by evoking those independent mechanisms, admixtures of LT-IIb(T13I) and LT-IIb-B5 will cooperatively augment Ag-specific immune responses to levels greater than are induced by use of either adjuvant alone. We will investigate: (i) the cooperative characteristics of admixtures on immunostimulation;(ii) the effects of LT-IIb(T13), LT-IIb-B5, and admixtures on activation and migration of DC from local lymphoid tissue to the draining lymph nodes; (iii) the roles of gangliosides in immunostimulation and HLT neurotrafficking, and (iv) the capacity of LTIIb( T13I), LT-IIb-B5, and admixtures as adjuvants to enhance protection against pathogens [Porphyromonas gingivalis, Mycobacterium tuberculosis, Streptococcus pneumoniae] which infect different compartments (oral, respiratory, systemic) in the host. With our long term experiences in studying type II HLTs, we are uniquely positioned to unravel the cellular &molecular events that underlie immunostimulation by HLTs and to evaluate the poorly understood roles of gangliosides in immunomodulation.